The mussel Mytilus edulis and the cultured ark shell Anadara broughtonii in the southeast coasts of the Seto Inland Sea were contaminated with paralytic shellfish poison (PSP) following the appearance of the dinoflagellate Alexandrium tamiyavanichii in early December 1999. A. tamiyavanichii plankton collected around the Straits of Naruto on December 3, 1999 showed PSP toxicity, of which 83 mol% was accounted for by GTX2, GTX3 and GTX4. Its specific toxicity was 112.5 fmol/cell, and one MU was equivalent to 7,200 cells. Toxicity values at the beginning of toxification were 4.7 MU/g for the ark shell and 7.3 MU/g for the mussel. In the former, the value remained at almost 4 MU/g, resulting in prohibition of marketing for about two months. In the latter, it sharply decreased to less than 4 MU/g. These bivalves collected during the toxification period were dissected into five tissues, mantle, adductor muscle, hepatopancreas, gills and “others”, and submitted to high-performance liquid chromatography (HPLC). The cultured ark shell accumulated GTX2, GTX3 and STX as major components and GTX1, GTX4, GTX5, neoSTX, dcSTX and PX1-3 (C1-C3) as minor ones. The amount of GTX3 decreased with time, while STX tended to increase. At the early stage of PSP toxification, toxins were accumulated in the gills and “others”, most of which were quickly detoxified. On the other hand, PSP of the toxified mussel consisted of GTX4 as a main component, and GTX1, GTX2, GTX3, GTX5, STX and PX1-2 (C1-C2) as minor ones. Its toxin composition pattern was similar to that of the ingested causative plankton. Its total toxin decreased soon after disappearance of the dinoflagellate. During the decrease of toxicity, PSP tended to be retained in the hepatopancreas, resulting in accumulation of 50 mol% of total toxin.
A method is described for the determination of the anthelmintic levamisole in muscle, liver, kidney and fat of cattle, swine and poultry using high performance liquid chromatography with photodiode array detection. Levamisole was extracted from an alkaline sample with ethyl acetate and back-extracted with 0.1 mol/L hydrochloric acid. The extract was applied to an SCX solid-phase extraction column. The column was washed with water and methanol. Levamisole was eluted with a solution of ammonia in methanol. The eluate was evaporated to dryness and the residue was dissolved in the mobile phase and injected into the HPLC system. Mean recoveries from 0.01∼0.10 μg/g fortified muscle, liver, kidney and fat samples ranged from 78.3 to 99.8%. The detection limit for the assay was 0.005 μg/g.
Amounts of isothiocyanates and related compounds in a mustard extract and a horseradish extract for food additive use were determined by GC, after confirmation of the identity of GC peaks by GC/MS. Amounts of allyl isothiocyanate, which included that of allyl thiocyanate, because most of the allyl thiocyanate detected in the sample was assumed to have been formed from allyl isothiocyanate during GC analysis, were 97.6% and 85.4%, in the mustard extract and the horseradish extract, respectively. Total amounts of the identified isothiocyanates in the mustard extract and the horseradish extract were 98.5% and 95.4%, respectively. Allyl cyanide, a degradation product of allyl isothiocyanate, was found in the mustard extract and the horse-radish extract at the levels of 0.57% and 1.73%, respectively. β-Phenylethyl cyanide, a possible degradation product of β-phenylethyl isothiocyanate, and allyl sulfides were found in the horseradish extract, at the levels of 0.13% and 0.46%, respectively. Allylamine, which is another degradation product of allyl isothiocyanate, was determined after acetylation, and was found in the mustard extract and the horseradish extract at the levels of 8 μg/g and 67 μg/g, respectively.
Acid-stable carmine has recently been distributed in the U.S. market because of its good acid stability, but it is not permitted in Japan. We analyzed and determined the structure of the major pigment in acid-stable carmine, in order to establish an analytical method for it. Carminic acid was transformed into a different type of pigment, named acid-stable carmine, through amination when heated in ammonia solution. The features of the structure were clarified using a model compound, purpurin, in which the orientation of hydroxyl groups on the A ring of the anthraquinone skeleton is the same as that of carminic acid. By spectroscopic means and the synthesis of acid-stable carmine and purpurin derivatives, the structure of the major pigment in acid-stable carmine was established as 4-aminocarminic acid, a novel compound.
A detection method using polymerase chain reaction (PCR) was developed to detect the genetically modified (GM) potato (NewLeaf Plus® potato; NL-P), which has not been authorized as safe in foods in Japan. The potato sucrose synthase gene was used as an internal control. The DNA from NL-P specifically provided an amplified band using PCR with a primer pair recognizing PLRV-rep gene. In addition, to prevent false-positive results in processed potato foods infected with PLRV, we designed a primer pair recognizing sequences derived from two organisms to detect specifically NL-P in processed potato. The PCR product obtained using the designed primer pair was specific for NL-P. The DNA introduced into NL-P could be detected from potato powder samples containing 0.05% NL-P. The proposed method was applied to the detection of NL-P in 25 processed potato foods. NL-P was detected in 3 snack products.
A method was developed for the analysis of ethychlozate (CIE) and its decomposition compound, 5-chloro-3(1H)-indazolylacetic acid (CIA) in fruits by HPLC and LC/MS. The sample was homogenized with 1 mol/L HCl, and CIE and CIA were extracted with 5 mol/L HCl and acetone. They were extracted from the acetone extract with diethylether-n-hexane (2 : 1). CIE was hydrolyzed to CIA with methanol-4 mol/L KOH (1 : 1). The solution was made acidic, and CIA was extracted with diethylether-n-hexane (2 : 1). The extract was cleaned up on a silica gel column. CIA was determined by HPLC-UV and LC/MS (Scan or SIR). Four fruits were spiked with CIE or CIA at 0.5 μg/g and analyzed by the proposed method with HPLC. The average recoveries were 77.2∼83.2% for CIE and 71.2∼89.2% for CIA. The concentrations determined by LC/MS were 10∼25% higher than the values by HPLC. The limit of detection (LOD) of CIA standard solution by HPLC corresponds to 0.015 μg/g of CIE in the sample. In the same way, the LOD of CIA by LC/MS (SIR) corresponds to 0.009 μg/g of CIE in the sample.
A simple method using Florisil cartridges was developed for the determination of dimethylformamide (DMF) in sucrose esters of fatty acids present in sugar esters (SuE) and sucrose acetate isobutyrate (SAIB) used as food additives. SuE was dissolved in acetone and loaded on a Florisil cartridge. SAIB was dissolved in hexane, loaded on a Florisil cartridge and washed with 10% acetone in hexane. The columns were eluted with acetone and DMF in the eluates was determined by GC with an FID detector. Recoveries of DMF at the level of 0.5∼100 μg/g were 93.3∼102.6%. The determination limit was 0.5 μg/g.
Determination of K-value (fish freshness index) and histamine (Hm) of mackerel and tuna during storage at various temperatures was done by using the oxygen-sensor method. Hm in mackerel increased even at low temperature (5°C), and reached the Defect Action Level (DAL) of the U.S. FDA, 5 mg/100 g, before the K-value approached the inedible level. As the storage temperature was increased, the rate of Hm formation increased remarkably and the amount of Hm in fish meat passed through the DAL, and reached the real hazard level, AL (50 mg/100 g), in a short time. The initial Hm level of raw tuna was too low to determine by our oxygen-sensor method, and the final Hm level after 8 days' storage at under 5°C was only 0.6 mg/100 g. But, when the storage temperature was elevated, the rate of Hm formation increased rapidly, as in mackerel, and the Hm level reached the DAL, then the AL, in a short time. Simultaneous determination of K-value and Hm is recommended for accurate and reliable quality inspection of fish and fish products.
A simple and rapid method for spore rec-assay by utilizing dry sheet medium culture (Compactdry TC, CTC) for determining numbers of bacteria, instead of the spore agar plate, was developed. One mL of spore suspension (2×106/mL) of Bacillus subtilis strain M45 Rec- or H17Rec+ was inoculated in the center of the CTC plate. In the case of metabolic activation, 1 mL of mixed solution (spore suspension of M45 or H17: 9,000×g supernatant of rat-liver homogenate treated with Aroclor 1254=19 : 1) was used. The spore suspension spreads over the whole sheet in seconds and gels. A paper disk impregnated with 20∼40 μL of the sample solution and 20 μL of the cofactor solution was placed on the surface of CTC plate. For the assay of samples that do not require metabolic activation, use of the cofactor solution can be omitted. After 48 hr incubation at 37°C, 0.01% MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] aqueous solution (0.5 mL) was dropped uniformly on the plate. The plate was left for 5 min, and the diameter of the inhibition circle was measured with slide calipers. The samples for which the difference in inhibition zone between M45 and H17 was more than 2 mm were judged positive. Under these conditions, the DNA damaging activities of sodium sulfite, sodium benzoate and citric acid, used as food additives, were investigated by the proposed method. Sodium sulfite and sodium benzoate gave positive results and citric acid gave a negative result with or without metabolic activation, in agreement with the results obtained by the conventional method.
The mean concentrations and daily intake of four antifungal agents were estimated based on the results of an analysis of 7,005 samples of food obtained in official inspections by Japanese local governments in fiscal year 1998. The mean concentration of diphenyl was 0.0004% of the allowable limit, and those of imazalil, o-phenylphenol, and thiabendazole were 14.0%, 3.5%, and 5.7%, respectively. The daily intakes of these antifungal agents per person, estimated from their concentrations and the daily consumption of the foods, were 0.000326, 1.89, 11.5, and 23.3 μg, respectively, and assuming a body weight of 50 kg, the amounts of these antifungal agents consumed were 0.000013%, 0.15%, 0.12%, and 0.47% of the acceptable daily intake, respectively. These values are similar to the values obtained on the basis of the results of the official inspection in fiscal years 1994 and 1996, except that the amount of diphenyl is much lower (1/100).